Critical issues in cancer immunotherapy are readily apparent in melanoma and include the findings that: I) melanomas grow in patients in spite of being infiltrated with lymphocytes; II) lymphocytes that lyse melanoma targets in vitro often do not demonstrate therapeutic effects after adoptive transfer to patients in vivo; and III) induction of cytolytic T lymphocytes (CTL) in melanoma patients with CTL-defined antigens (Ag)/peptides did not correlate with clinical responses. Understanding the mechanisms underlying these phenomena holds the promise of revolutionizing CTL-based immunotherapies. However, these mechanisms can not be addressed directly in vivo in patients and thus far relevant in vitro models have been lacking. In the proposed studies our major goal is to use the human melanoma organotypic (dermis reconstruct) culture model to develop novel CTL-based adoptive and active immunotherapies against melanoma. Our preliminary studies have demonstrated that CTL which were generated against melanoma cells in a mixed lymphocyte/tumor cell culture (MLTC) are able to lyse the melanoma cells in the human melanoma/dermis reconstruct at a reduced effector-to-target (E:T) cell ratio. Our Specific Aims are to: 1) Determine in vitro in the reconstruct model the factors that play a role in the activation or inhibition of CTL activity against melanoma cells in vivo in patients. Identification of these factors will reveal new ways to make effective adoptive immunotherapy of cancer patients. 2) Evaluate the potential of the human dermis reconstruct model for the generation of new CTL from peripheral blood mononuclear cells (PBMC) or tumor-infiltrating lymphocytes using cultured or fresh melanoma cells as stimulants and compare the CTL activity generated in this model with that generated in MLTC. These studies are likely to isolate the ?optimal? CTL which has been induced (committed) under in vivo-like conditions eliminating the artifacts of in vitro culture systems. 3) Clone selected Ag recognized by the CTL that are highly effective in the reconstruct model with emphasis on the CTL induced against fresh melanoma specimens. The Ag recognized by optimally effective CTL identified in the reconstruct will be cloned using the well established COS cell system. 4) Design and implement clinical trials with adoptively transferred CTL and CTL-defined Ag in melanoma patients. The proposed studies are translational, based on the development of a novel human organotypic culture offering novel immunotherapies of melanomas using adoptive transfer of CTL or CTL-defined vaccines.